The invention relates generally to novel compositions and methods for detecting the presence of viruses that frequently infect humans and are associated with the development of human disease. More particularly, the invention is directed to an accurate and sensitive method for the diagnosis and quantitation of Epstein-Barr virus infection using specific oligonucleotides as primers to amplify particular regions of the genome of the virus sought to be detected in a clinical specimen. Epstein-Barr virus-specific oligonucleotides may be used in the subsequent detection of the amplified regions of DNA.
Epstein-Barr virus (EBV), a human herpes virus, is ubiquitous in humans. Antibodies to polypeptides of the virus are present in over 80% of human serum samples from the United States and in even higher percentages from populations in Asia and Africa. Although it is prevalent throughout the world, the consequences of EBV infection vary among different populations. The virus is responsible for of infectious mononucleosis, a benign proliferation of infected B-lymphocytes, in Western countries and is implicated in Burkitt""s lymphoma in Africa and nasopharyngeal carcinoma (NPC) in Asia. EBV can also cause acute and rapidly progressive B lymphoproliferative disease in severely immune compromised patients.
As a result of the immunosuppression necessary to maintain the function of transplanted organs, transplant patients are all at risk for developing EBV infection and therefore post transplant lymphoproliferative disorder (PTLD). However, the group at highest risk for this complication is the liver transplant population. This is because these patients are generally very young, frequently less than 5 years of age, and therefore they frequently have not yet been exposed to EBV and as a result do not have a natural immunity to the virus.
Approximately 50% of the transplanted population develop EBV infection during the post-transplant course, frequently symptomatic including PTLD. These patients therefore are a prime population for monitoring the development of EBV infection and its associated complications. Approximately 50% of these patients will develop EBV infection requiring monitoring of the peripheral blood EBV DNA levels.
Nationally there are between 450-500 liver transplants in pediatric patients. In those patients under 14 months of age approximately 75% of them develop EBV infections. These numbers do not include all the bone marrow, kidney, heart, and lung transplants in pediatric patients. And although there is a lower incidence of EBV lymphoproliferative complication in all of the adult transplant patients, there is a clinically significant incidence of this complication in these patients as well. The demand for this kind of patient monitoring therefore is high. Currently, there are a few recognized labs who provide quantitative EBV analyses.
EBV associated post-transplant lymphoproliferative disease (PTLD) is a major cause of morbidity and mortality for children and adults who undergo solid organ transplantation and bone marrow transplantation (Ho M, et al. The frequency of Epstein-Barr virus infection and associated lymphoproliferative syndrome after transplantation and its manifestations in children. Transplantation 45:719-727, 1988).
Patients who are EBV naxc3xafve and receive an organ from an EBV positive donor, especially those treated with anti-T-cell immunotherapy (anti-thymocyte globulin or monoclonal antibodies), are at high risk for developing PTLD. Infants and toddlers, who comprise 50% of the pediatric liver transplant population, are usually EBV naxc3xafve. More than 75% of patients at risk acquire the virus within the first year of life. Up to 15% of liver transplant recipients who are at high risk will develop PTLD. Consequently, the risk of infection and complication is substantial.
For children, especially those less than two years of age, PTLD is a critical factor affecting cost, graft function and quality of life. Attempts to treat these transplant patients with interferon, acyclovir, and anti-B cell monoclonal antibodies are generally not successful once an EBV infection has been established or reactivated (Papadopoulos E B, et al. Infusions of donor leukocytes as treatment of Epstein-Barr virus associated lymphoproliferative disorder complicating allogeneic marrow transplantation. N Engl J Med 330: 1185, 1994; Zutter M M, et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood 72, 520, 1988; Shapiro R S, et al. Epstein-Barr virus associated B-cell lymphoproliferative disorders following bone marrow transplantation. Blood 71: 1234, 1988; Antin J et al. Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: Effective prophylaxis for graft-vs-host disease in patients with hematologic malignancies. Blood: 78: 2139, 1991; Martin P J, et al. Fatal Epstein-Barr virus associated proliferation of donor B cells after treatment of acute graft vs-host disease with a murine anti-T-cell antibody. Ann Intern Med 101:310, 1984). A number of studies however have shown that PTLD may be reversible in solid-organ transplant recipients following reduction or discontinuation of immune suppression (Starzl T, et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin steroid therapy. Lancet I: 583, 1984).
Differences in levels of EBV DNA found in the peripheral blood post-transplant may distinguish which patients are at highest risk to develop EBV PTLD and therefore could permit earlier intervention in these patients. Previous studies have shown a correlation between levels of EBV DNA and the occurrence of EBV-PLTD in organ transplant patients (Kenagy D N, et al. Epstein-Barr virus DNA in peripheral blood leukocytes with post-transplant lymphoproliferative disease. Transplant 60: 547, 1995; Riddler S A, et al. Increased levels of circulating Epstein-Barr virus (EBV) infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of post-transplant lymphoproliferative disease in solid-organ transplant recipients Blood 84: 972, 1994; Savoie A, et al. Direct correlation between the load of Epstein-Barr virus infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 83: 2715, 1994). These results suggest the potential benefit of monitoring EBV DNA levels in the peripheral blood of the transplant patients may be clinically useful in the management and possible preemptive intervention of the development of PTLD in these patients. As a result, a number of assays using a variety of approaches have been developed and reported for either semiquantitative or quantitative determination of EBV DNA levels.
Most of the semiquantitative assays are based on standard PCR analysis of a dilution series of patient samples compared to a known standard. While extremely useful, quantitative PCR can be very laborious to perform. Most of the difficulties arise because only a very small number of the cycles in a PCR reaction contain useful information. The early cycles have undetectable amounts of the DNA product and late cycles (plateau phase) are almost as uninformative. The PCR product is then detected by gel electrophoresis followed by ethidium bromide staining of the gel. The result is determined by the lowest dilution at which a band is visible (Lucas K G, et al. Semiquantitative Epstein-Barr virus polymerase chain reaction for the determination of patients at risk for EBV induced lymphoproliferative disease after stem cell transplantation. Blood 91: 3654, 1998; Baldanti F, et al. High levels of Epstein-Barr virus DNA in blood of solid organ transplant recipients and their value in predicting post-transplant lymphoproliferative disorders. J. Clin Microbiol. 38: 613, 2000; Rogers B, et al. Epstein-Barr virus polymerase chain reaction and serology in pediatric post-transplant lymphoproliferative disorder: three year experience. Pediatric and Developmental Pathology 1: 480, 1998). The quantification of the PCR and RT-PCR products is therefore based on an endpoint approach rather than using a kinetic approach. Since some labs use probe specific confirmation of their PCR products and others do not, the specificity and sensitivity of these assays are quite variable.
The standards used for the quantitative determinations also vary, and even the sample preparation differs as some assays base the results on the number of copies per ml of whole blood, others use the number of isolated white blood cells as the basis, while still other labs base the determination on extracted total DNA quantity.
More recently the introduction of xe2x80x9creal-timexe2x80x9d quantitative PCR analysis has been developed for the determination of EBV viral DNA levels (Niesters H et al. Development of a real-time quantitative assay for detection of Epstein-Barr virus. J Clin Microbiol. February 2000; 38(2):712-5). Real-time or kinetic PCR is a powerful method for determining the initial template copy number. The quantitative information in a PCR reaction comes from the few cycles where the amount of DNA grows logarithmically from barely above background to the plateau. Often only 6 to 8 cycles out of 40 will fall in this log-linear portion of the curve. Since the fluorescence signal is acquired during each cycle, data from the critical cycles can be captured, quantified and the fluorescence plotted against the cycle number.
This technology has improved the dynamic range in which samples can be analyzed quantitatively without dilution which reduces substantially the turnaround time and is much less labor intensive and less prone to technical errors due to less manipulation involved (Kimura H, et al. Quantitative analysis of Epstein-Barr virus load by using a real-time PCR assay. J. Clin Microbiol. 37:132, 1999; Martell M, et al. High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA J Clin Microbiol. February 1999; 37(2):327-32; Mercier B, et al. Simultaneous screening for HBV DNA and HCV RNA genomes in blood donations using a novel TaqMan PCR assay. J Virol Methods. January 1999; 77(1):1-9). This latter approach provides the best opportunity to provide routine monitoring of the transplant patient population for early evidence of EBV infection. Early detection and recognition of patients at high risk for PTLD will provide opportunities for preemptive therapy rather than treating and managing the complications of advanced PTLD.
Therefore, it would be advantageous to have an accurate and sensitive method for the diagnosis and quantitation of Epstein-Barr virus infection in a clinical specimen.
It is an object of the present invention to disclose a nucleic acid sequences (oligonucleotides) useful as primers and/or probes in the detection of a Epstein-Barr virus in clinical specimens. Also, the present invention is directed to a method of detecting the presence of Epstein-Barr virus in a clinical specimen wherein the oligonucleotides of the present invention may be used to amplify target nucleic acid sequences of a Epstein-Barr virus that may be contained within a clinical specimen, and/or to detect the presence or absence of amplified target nucleic acid sequences of the Epstein-Barr virus. Respective oligonucleotides may be used to amplify and/or detect EBV and EBV nucleic acid sequences. By using the oligonucleotides of the present invention and according to the methods of the present invention, as few as one to ten copies of the Epstein-Barr virus genome may be detected.
One object of the present invention is to provide oligonucleotides that can be used as primers to amplify specific nucleic acid sequences of EBV.
Another object of the present invention is to provide oligonucleotides that can be used as probes in the detection of amplified specific nucleic acid sequences of EBV.
Among the nucleic acids provided herein are the nucleic acids whose sequence is provided in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 OR SEQ ID NO:8, or a fragment thereof. Additionally, the invention includes mutant or variant nucleic acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or a fragment thereof, any of whose bases may be changed from the corresponding bases shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, while still hybridizing to an EBV DNA sequence. The invention further includes the complement of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, including fragments, derivatives, analogs and homologs thereof. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
The invention also includes an oligonucleotide that includes a portion of the disclosed nucleic acids. For example, the oligonucleotide can be at least 10 nucleotides in length and include at least nine contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
A further object of the present invention is to provide an accurate and sensitive method for detecting the presence of EBV that may be contained in clinical specimens by using the oligonucleotides disclosed to amplify and detect specific nucleic acid sequences of EBV.
Another object of the present invention is to provide oligonucleotides that can be used as primers to amplify specific nucleic acid sequences of EBV.
Another object of the present invention is to provide oligonucleotides that can be used as probes in the detection of amplified specific nucleic acid sequences of EBV.
Another object of the present invention is to provide oligonucleotides that can be used as primers to amplify DNA sequences from the EBNA2 region of the EBV genome.
A further object of the present invention is to provide an accurate and sensitive method for detecting the presence of EBV that may be contained in clinical specimens by using the oligonucleotides disclosed to amplify and detect specific nucleic acid sequences of EBV.
It is a further object of the invention to provide a kit for identifying or amplifying a gene encoding an Epstein-Barr virus polypeptide.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.